Triazine carboxylic acids and esters
BACKGROUND OF THE INVENTION 
     This invention generally pertains to heterocyclic carbon compounds having drug and bio-affecting properties and to their preparation and use. In particular, the invention is concerned with various heterocyclic derivatives characterized by Formulas I and II, infra., which are inhibitors of blood platelet aggregation. 
     United States patents relating to the carboxylic acid and ester derivatives of the various heterocycles disclosed herein are as follows: 
     Triazine compounds in Field of Search 514/242 and 182. 
     Pitet, et al., U.S. Pat. No. 4,167,566 discloses 2-phenyl-3-oxo-5,6-diaryl-AS-triazines having antithrombogenic properties of formula (1). ##STR2## 
     Lacefield, U.S. Pat. No. 4,318,911 discloses 5,6-diaryl-1,2,4-triazines having antithrombogenic activity of the formula (2). ##STR3## 
     Pitet, et al., U.S. Pat. No. 4,511,566 discloses 2-N-cycloalkylmethyl-3-oxo-5,6-diaryl-as-triazines having analgesic activity of formula (3). ##STR4## 
     Guy, et al., U.S. Pat. No. 4,677,105 discloses 2-alkoxycarbonylalky-3-oxo-5,6-diaryl-as-triazines having non-inflammatory analgesic activity of formula (4). ##STR5## 
     Pitit, et al. U.S. Pat. No. 4,851,411 discloses 5-monoaryl as-triazin-3-ones having antianxiety or antidepressant activity of formula (5). ##STR6## 
     SUMMARY OF THE INVENTION 
     In its broadest aspect, this invention is concerned with heterocyclic carboxylic acids and esters of Formula I and Formula II ##STR7## wherein HET 1 , HET 2 , R, R 1 , and n are defined below which are inhibitors of adenosine diphosphate and collagen-induced aggregation of human platelet-rich plasma and are particularly useful as inhibitors of mammalian blood platelet aggregation. 
     Another embodiment of the invention relates to the alkali metal salts of carboxylic acids of Formula I (R is hydrogen) and Formula II (R 1  is hydrogen). A further embodiment concerns pharmaceutical compositions comprised of a Formula I or II compound combined with at least one pharmaceutically acceptable excipient. Yet another embodiment relates to a method for inhibiting blood platelet aggregation in a mammal which comprises administering a therapeutically effective amount of a compound of Formula I or an alkali metal salt thereof where R is hydrogen or a Formula II compound or an alkali metal salt thereof where R 1  is hydrogen to a mammal in need of such treatment. 
     DETAILED DESCRIPTION OF THE INVENTION 
     This invention relates to inhibitors of mammalian blood platelet aggregation of Formula I 
     
         HET.sub.1 -(CH.sub.2).sub.n CO.sub.2 R                     (I) 
    
     wherein n is 6-9, R is hydrogen, lower alkyl or an alkali metal ion, and HET 1  is the heterocyclic radical ##STR8## 2,3-dihydro-3-oxo-5,6-diphenyl-1,2,4-triazin-2-yl. 
     The compounds of the instant invention are further characterized by Formula II ##STR9## wherein 
     R 1  is hydrogen, lower alkyl or an alkali metal ion, and the radical --OCH 2  CO 2  R 1  is attached in the 3 or 4 ring position; 
     HET 2  is the heterocyclic radical ##STR10## 2,3-dihydro-3-oxo-5,6-diphenyl-1,2,4-triazin-2-yl. 
     It is understood that as used herein limitations of Formula I and II are defined as follows. 
     The term &#34;lower alkyl&#34; refers to a branched or unbranched saturated hydrocarbon chain containing from 1-4 carbon atoms: specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl, secondary butyl, and tertiary butyl. 
     The term &#34;lower alkanol&#34; denotes an alcohol having 1-4 carbon atoms defined by &#34;lower alkyl&#34;. 
     The symbol &#34;Ph&#34; represents phenyl. 
     The term &#34;alkali metal ion&#34; refers to ions derived from the alkali metals, most preferably sodium and potassium. 
     According to the present invention the compounds characterized by Formula I are obtained by a process comprising: 
     (a) hydrolyzing a compound of Formula (I a ) 
     
         HET.sub.1 --(CH.sub.2).sub.n CO.sub.2 R.sup.a              (I.sup.a) 
    
     wherein HET 1  is as defined above, n is 6-9 and R a  is lower alkyl, or 
     (b) esterifying a compound of Formula (I b ) 
     
         HET.sub.1 --(CH.sub.2).sub.n CO.sub.2 H                    (I.sup.b) 
    
     wherein HET 1  is as defined above and n is 6-9 with a lower alkanol, or 
     (c) alkylating HET 1  -H wherein HET 1  is as defined above with a compound of Formula (III) 
     
         X--(CH.sub.2).sub.n CO.sub.2 R.sup.a                       (III) 
    
     wherein X is halogen, preferably bromine,, n is 6 to 9, and R a  is lower alkyl. 
     Scheme 1 below illustrates the foregoing process. ##STR11## 
     Compounds of Formula I are conventionally prepared as shown in Scheme 1 by base-mediated alkylation of the parent heterocycle (III) with a lower alkanol ester of an omega-halogenalkanoic acid (IV) to provide esters (I a ). Alkylation can be carried out with sodium hydride, potassium hydride, potassium t-butoxide in suitable solvents such as tetrahydrofuran and dimethylformamide with conditions dependent upon the identity of the heterocycle and the acidity of the proton being replaced. Preferred alkylating conditions employ sodium hydride in dimethylformamide (DMF) at room temperature or potassium carbonate in DMF at 110° C. In those instances where the alkylation results in mixtures of regioisomers, separation is readily accomplished by chromatography on silica gel. Structural assignments are made from consideration of the  1  H and  13  C NMR spectra. The heterocycles (III) employed as starting material are synthesized by methods reported in the chemical literature or by minor modifications thereof readily apparent to a skilled chemist. 
     Esters (I a ) are converted to the corresponding acids (I b ) under the influence of either aqueous alkali or aqueous acid depending upon the sensitivity of the heterocycle. Conversely, acids (I b ) are conventionally converted to the corresponding esters (I a ) by heating the acid in a lower alkanol in the presence of an inorganic acid such as hydrochloric, sulfuric and the like. 
     Alkali metal salts of Formula I carboxylic acids (R is an alkali metal ion) are conventionally prepared by dissolving the acid in a suitable solvent such as methanol, adding a molar equivalent of an alkali base such as sodium methoxide, and precipitating the salt or removing the solvent. 
     According to the present invention, the compounds characterized by Formula II are obtained by a process comprising: 
     (a) hydrolyzing a compound of Formula II a  ##STR12## wherein Het 2  is as defined above and R 1   a  is lower alkyl; or 
     (b) esterifying a compound of Formula II b  ##STR13## wherein HET 2  is as defined above; or 
     (c) alkylating HET 2  -H wherein HET2 is as defined above with a compound of Formula (V) ##STR14## wherein R 1   a  is lower alkyl and X is halogen or an aryl sulfonate; or 
     (d) alkylating a compound of Formula (VI) ##STR15## wherein HET 2  is as defined above with BrCH 2  CO2 2  R 1   a  is lower alkyl. 
     The following scheme for preparation of representative compounds of Formula II illustrates the foregoing process. ##STR16## 
     Scheme 2 depicts several different approaches for synthesis of compounds incorporating the aryloxyacetic acid moiety. Direct alkylation of heterocycle (1) with either bromide (2) or tosylate (TS) (3) available from alcohol (5), was accomplished with sodium hydride in DMF at room temperature or potassium carbonate in DMF at 110° C to furnish esters (6). Alternatively, in some cases, reaction of the heterocycle (1) with alcohol (5) obtained from diol (4) in the presence of diethyl azodicarboxylate and triphenylphosphine provided esters (6) under the mild conditions characteristic of the Mitsunobu reaction, Synthesis. 1-28 (1981). Carboxylic acids (7) were obtained by alkaline hydrolysis of esters (6). 
     Alkali metal salts of Formula II carboxylic acids (R 1  is an alkali metal ion) are conventionally prepared by dissolving the acid in a suitable solvent such as methanol, adding a molar equivalent of an alkali base such as sodium methoxide, and precipitating the salt or removing the solvent. 
     As stated above, the compounds of Formula I and Formula II have pharmacological properties which make them particularly useful as inhibitors of blood platelet aggregation. 
     Platelet aggregation is considered part of a complex physiological mechanism for formation of a thrombus in the vascular system. Thromboembolic phenomena, i.e., the formation of thrombi, are involved in hemostasis and a number of disease states in mammals including thrombophlebitis, phlebothrombosis, cerebral thrombosis, coronary thrombosis and retinal vessel thrombosis. An increase in propensity for platelet aggregation, sometimes referred to as platelet adhesiveness, is observed following parturition, surgical operations such as coronary artery bypass surgery, organ transplant, angioplasty, prosthetic heart valve implants to name a few and in ischaemic heart disease, artherosclerosis, multiple sclerosis, intracranial tumors, thromboembolism, and hyperlipemia; refer to A. Poplawski, et al., J. Artherosclerosis Research, 8, 721 (1968). Thus, the compounds of the invention which have antithrombogenic actions (inhibit blood platelet aggregation) are useful in prevention or treatment of conditions involving platelet aggregation and thrombosis such as the above. The instant compounds are also considered to have antimetastatic potential in view of their platelet inhibition properties. 
     The pharmacological properties of the instant compounds can be demonstrated by conventional in vitro and in vivo biological tests such as the following. 
     IN VITRO INHIBITION OF HUMAN PLATELET AGGREGATION 
     The aggregometer method of Born, C.V.R., J. Physiol., (London), 1962, 162, 67-68, as modified by Mustard, J.F., et al., J. Lab. Clin. Med. 1964, 64, 548-599 was used to assess the in vitro activity of the various compounds as to the inhibition of adenosine diphosphate (ADP) and collagen-induced platelet aggregation. The human volunteer donor&#39;s arm is cleansed with 70% ethyl alcohol. A sterile 20 ml syringe and needle are used to withdraw 20 ml of blood. The blood is immediately added to a test tube containing 3.8% sodium citrate to prevent clotting (1 part citrate to 9 parts blood). 
     Platelet rich plasma (PRP) was separated by centrifugation for 10 minutes at 1000 rpm (140xg) from citrated (3.8%) human blood. All glassware used for preparation of PRP is silicon treated. ADP in final concentration of 0.5 mcg/mL or 0.05 mL of a collagen suspension prepared according to the method described by Evans, G., et al., J. Exp. Med., 1968, 128, 877-894 was used to induce aggregation. The various compounds tested were dissolved in dimethylsulfoxide (DMSO) so that 5 mcl added to the platelet rich plasma would yield the desired test concentration. Vehicle control trials were done and compared with aggregation induced in platelet rich plasma containing various concentrations of the test compounds. Dose response curves were obtained and Inhibitor Concentration (IC 50 ) values calculated. In this test, the IC 50  values for dipyridamole, a clinically useful antithrombogenic agent, are 512 mcg/ml vs. ADP and 245 mcg/ml vs collagen. Results for 50% inhibition of ADP-induced aggregation are given in Tables I and II hereinafter for Formula I and II compounds. 
     
                       TABLE 1                                                     
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Inhibition of Human Platelet Aggregation                                  
of Formula I Compounds (IC.sub.50 mcg/ml)                                 
HET.sub.1 --(CH.sub.2).sub.n CO.sub.2 R                                   
                                     vs. ADP                              
Example HET.sub.1        n      R    mcg/ml                               
______________________________________                                    
1       2,3-dihydro-3-oxo-5,6-                                            
                         8      CH.sub.3                                  
                                     6.8                                  
        diphenyl-1,2,4-triazin-2-yl                                       
2       2,3-dihydro-3-oxo-5,6-                                            
                         8      H    2.2                                  
        diphenyl-1,2,4-triazin-2-yl                                       
3       2,3-dihydro-3-oxo-5,6-                                            
                         7      CH.sub.3                                  
                                     4.6                                  
        diphenyl-1,2,4-triazin-2-yl                                       
4       2,3-dihydro-3-oxo-5,6-                                            
                         7      H    3.1                                  
        diphenyl-1,2,4-triazin-2-yl                                       
______________________________________                                    
 
    
     
                       TABLE 2                                                     
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Inhibition of Human Platelet Aggregation                                  
of Formula II Compounds (IC.sub.50 mcg/ml)                                
 ##STR17##                                                                
                                   Vs ADP                                 
Example                                                                   
       HET.sub.2           R.sub.1 mcg/ml                                 
______________________________________                                    
5      2,3-dihydro-3-oxo-5,6-diphenyl-                                    
                           CH.sub.3                                       
                                   1.0                                    
       1,2,4-triazin-2-yl                                                 
6      2,3-dihydrin-3-oxo-5,6-                                            
                           H       0.9                                    
       1,2,4-triazin-2-yl                                                 
______________________________________                                    
 
    
     The acids are particularly potent inhibitors of ADP-induced aggregation of human platelets. While the esters are generally less active than the parent acid, they are useful as pro-drugs in vivo where they are hydrolyzed to the corresponding acid. 
     The dosage employed in the therapeutic methods of the instant invention will vary with the form of administration, the particular compound chosen, the subject being tested and the effect desired. Suitable effective doses in animals range from 0.1-50 mg/kg body weight orally and from 0.05-10 mg/kg body weight parenterally (generally characterized as subcutaneous, intramuscular, and intravenous injection). It is contemplated that the effective unit dose in man will range from 1 to 100 mg and preferably from 1 to 20 mg administered one to three times a day. In accordance with conventional clinical practice, the effective dose can be determined by administering a Formula I or II compound at a dosage substantially less than the dose of the compound which is thought to be effective and then increasing the dosage in small increments until the desired effect is achieved. 
     In carrying out the instant therapeutic methods, the active ingredient of Formula I or II or alkali metal salts of Formula I and II carboxylic acids are preferably administered with a pharmaceutically acceptable carrier and such compositions constitute part of the instant invention. Suitable dosage forms for oral use are tablets, dispersible powders, granules, capsules, syrups and elixirs. Examples of parenteral forms are solutions, suspension, dispersions, emulsions, and the like. The compositions for oral use may contain one or more conventional adjuvants, such as sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a composition of suitable pharmaceutical elegance. Tablets may contain the active ingredient in admixture with conventional pharmaceutical acceptable excipients including inert diluents such as calcium carbonate, sodium carbonate, lactose and talc; granulating and disintegrating agents such as starch and alginic acid; binding agents such as starch, gelatin and acacia and lubricating agents such as magnesium stearate, stearic acid and talc. The tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. Similarly, suspension, syrups and elixirs may contain the active ingredient in admixture with any of the conventional excipients utilized for the preparation of such compositions such as suspending agents (e.g., methylcellulose, tragacanth, and sodium alginate), wetting agents (e.g., lecithin, polyoxyethylene stearate) and preservatives such as ethyl-p-hydroxybenzoate. Capsules may contain the active ingredient alone or admixed with an inert solid diluent such as calcium carbonate, calcium phosphate and kaolin. The injectible compositions are formulated as known in the art and may contain appropriate dispersing or wetting agents and suspending agents identical or similar to those mentioned above. 
     The following examples are given by way of illustration and are not to be construed as limiting the invention in any way inasmuch as many variations of the invention are possible within the spirit of the invention. 
    
    
     DESCRIPTION OF SPECIFIC EMBODIMENTS 
     In the following examples, all temperatures are given in degrees Centigrade. Melting points were recorded on a Thomas-Hoover capillar melting point apparatus and are uncorrected. Proton magnetic resonance ( 1  H-NMR) spectra were recorded on a Bruker AM 300, Bruker WM 360 or Varian Gemini 300 spectrometer. All spectra were determined in CDC1 3  or DMSO-d 6  unless otherwise indicated and chemical shifts are reported in delta units downfield from the internal standard tetramethylsilane (TMS) and interproton coupling constants are reported in Hertz (Hz). Splitting patterns are designated as follows: s, singlet: d, doublet: t, triplet: q, quartet: m, multiplet: br, broad peak: and dd, doublet of doublet. 
     EXAMPLE 1 
     Methyl 3-oxo-5,6-diphenyl-1,2,4-triazine-2(3H)-nonanoate ##STR18## 
     Sodium hydride (577mg of a 60% dispersion in oil, 14mmol) was washed twice with hexanes, covered with DMF (40mL) and 5,6-diphenyl-1,2,4-triazol-3(2H)-one (3g, 12mmol), obtained according to M. Friedman, J. Org. Chem., 30, 859-863 (1965), added. After gas evolution ceased, the mixture was stirred for 15 minutes before adding methyl 9-bromononanoate (3.32g, 13mmol). The mixture was stirred at room temperature for 15 minutes, before being poured onto water and extracted with diethyl ether (3x). The combined extracts were washed with water (3x), dried over sodium sulfate and concentrated to leave an oil which was chromatographed on a column of silica gel. Elution with a mixture of diethyl ether and hexanes (3:2) gave methyl-3-oxo-5,6-diphenyl-1,2,4-triazin-2(3H)-nonanoate (3.09g, 61%). 
     Anal. Calcd. for C 25  H 29  N 3  O 3  : C, 71.58; H, 6.97; N, 10.02. Found: C, 71.59: H, 7.13; N, 10.06%. 
       1  H-NMR (CDC1 3 ) delta: 1.20 to 1.45 (8H, m), 1.58 (2H, quintet, J=6Hz), 1.90 (2H, quintet, J=6Hz), 2.26 (2H, t, J=6Hz), 3.63 (3H, s), 4.21 (2H, t, J=6Hz) and 7.20 to 7.55 (10H, m). 
     EXAMPLE 2 
     3-Oxo-5,6-diphenyl-1,2,4-triazine-2(3H)-nonanoic acid ##STR19## 
     A mixture of methyl-3-oxo-5,6-diphenyl-1,2,4-triazine-2(3H)-nonanoate (1.90g&#39; 4.5mmol), 5N N (2.72mL, 13mmol) and methanol (30mL) was heated on a steam bath for 30 minutes. The volatiles were removed in vacuo and the residue diluted with water and dilute HC1 solution until pH=1 The mixture was extracted with CH 2  C1 2 , the combined extracts dried over sodium sulfate and concentrated to give an oil. Chromatography on a column of silica gel using diethyl ether as eluent gave 3-oxo-5,6-diphenyl-1,2,4-triazine-2(lH)-nonanoic acid (1.50g, 81%) which crystallized on standing and had mp 97-100° C. 
     Anal. Calcd. for C 24  H 27  N 3  O 3  : C, 71.09; H, 6.71; N, 10.36. Found: C, 71.29; H, 6.97: N, 10.21%. 
       1  H-NMR (CDC1 3 ) delta: 1.20 to 1.50 (8H, m), 1.57 (2H, t, J=7Hz), 1.88 (2H, quintet, J=7Hz), 2.27 (2H, t, J=7.5Hz), 4.19 (2H, t, J=7Hz), 7.20 to 7.60 (10H, m) and 10.89 (1H, bs). 
     EXAMPLE 3 
     Methyl 3-Oxo-5,6-diphenyl-1,2,4-triazine-2(3H)-octanoate ##STR20## 
     Reaction of 5,6-diphenyl-1,2,4-triazol-3(2H)one and methyl 8-bromooctanoate according to the procedure of Example 1 provided the title compound as an oil. 
     Anal. Calcd. for C 24  H 27  N 3  O 3  : C, 71.09; H, 6.71; N, 10.36. Found: C, 71.05: H, 6.87; N, 10.41. 
     EXAMPLE 4 
     3-Oxo-5,6-diphenyl-1,2,4-triazin-2(3H)-octanoic Acid ##STR21## 
     Hydrolysis of methyl 3-oxo-5,6-diphenyl-1,2,4-triazine-2(3H)-octanoate according to the procedure of Example 2 provided the title compound, m.p. 111°-113°C. 
     Anal. Calcd. for C 23  H 25  N 3  O 3  : C, 70.57; H, 6.44; N, 10.73. Found; C, 70.35; H, 6.72; N, 10.33. 
     EXAMPLE 5 
     Methyl [3-[2-(2,3-dihydro-3-oxo-5,6-diphenyl-1,2,4-triazin-2-yl)ethyl]phenoxy]acetate ##STR22## 
     Diethyl azodicarboxylate (2.72g, 2.50mL, 16mmol) was added to a stirred solution of 5,6-diphenyl-1,2,4-triazol-3(2H)-one (3.00g, 12mmol), triphenylphosphine (4.11g, 16mmol) and methyl [3-(2-hydroxyethyl)phenoxy]acetate (2.78g, 13mmol) in dry THF (75mL) maintained at 0° C under an atmosphere of nitrogen. The ice bath was removed and the mixture stirred at room temperature for 3 hours. The solvent was evaporated and the residue chromatographed twice on columns of silica gel using a mixture of diethyl ether and hexanes (7:3) as eluent to give methyl -[2-(2,3-dihydro-3-oxo-5,6-diphenyl-1,2,4-triazin-2-yl)ethyl]phenoxy]acetate (6.40g, 90%) as a yellow foam. 
     Anal. Calcd. for C 26  H 23  N 3  O 4 .0.5 H 2  O: C, 69.33; H, 5.38: N, 9.33: H2O, 1.99. Found: C, 69.23: H, 5.19: N, 9.56: H 2  O, 0.15%. 
       1  H-NMR (CDC1 3 ) delta: 3.19 (2H, t, J=7.5Hz), 3.72 (3H, s), 4.45 (2H, t, J=7.5Hz), 4.56 (lH, s), 6.70 to 6.80 (2H, m), 6.88 (1H, d, J=7.5Hz), 7.10 to 7.50 (11H, m). 
     EXAMPLE 6 
     [3-[2-(2,3-Dihydro-3-oxo-5,6-diphenyl 
     1,2,4-triazin-2-yl)ethyl]phenoxy]acetic Acid ##STR23## 
     A mixture of methyl [3-[2-(2,3-dihydro-3-oxo-5,6-diphenyl-1,2,4-triazin-2-yl)ethyl]phenoxy]acetate (4.00g, 9mmol), 5N NaOH (3.70mL, 18mmol) and methanol (60mL) was heated to reflux on a steam bath. After 20 minutes, the methanol was evaporated, the residue diluted with water and acidified with 2N HCl solution. A yellow solid was filtered off and recrystallized from a mixture of hexanes and CH 2  C1 2  to give [3-[2-(2,3-dihydro-3-oxo-5,6-(diphenyl-1,2,4-triazin-2-yl)ethyl]phenoxy]acetic acid (2.79g, 72%), mp 179-182° C. 
     Anal. Calcd. for C 25  H 21  N 3  O 4 .0.5H 2  O: C, 68.80; H, 5.09; N, 9.63: H20, 2.06. Found: C, 68.81; H, 4.79; N, 8.64: H 2  O, 0.51%. 
       1  H-NMR (DMSO-d 6 ) delta: 3.10 (2H, t, J=7.5Hz), 4.36 (2H, t, J=7.5 Hz), 4.62 (2H, s), 6.70 to 6.95 (3H, m), 7.10 to 7.60 (11H, m) and 12.97 (1H, bs).